1. Technical Field
This invention relates to methods for the detection of analyte particles, and determining their interaction characteristics within complex media. More particularly, this invention relates to diffusion immunoassays and their functional equivalents.
2. Description of the Related Art
Biologically relevant molecules, such as proteins and nucleic acids, are commonly associated with biological systems, where they form a complex network of interactions for the performance of tasks such as cell replication, metabolism, self-regulation, intercellular signaling, and immune response. Diseases distort this network, and understanding this distortion is fundamental to early detection of disease and chemical repair of the distortion through drug therapy. There are a number of existing techniques for identifying and characterizing these large molecules to gain an understanding of the interaction network, but each suffers from particular limitations. Techniques used for nucleic acids, such as DNA, have been largely successful due to their analytically favorable properties, but proteins are chemically and physically diverse. This diversity results in analytical techniques that are by necessity narrowly focused, when a broad technique would be much more helpful in characterizing a complex protein network. Furthermore, proteins may be functionally significant at even undetectable concentrations, yet cannot be amplified with the ease of nucleic acids, necessitating techniques that have a high intrinsic sensitivity.
Genetic Methods
Protein interactions can be investigated by using classical genetics. Different mutations are combined into in the same cell or organism, and then the resulting phenotype is observed. This ensures that protein interactions occur in their near perfectly native environments, Unfortunately, these methods are applicable only to a small group of proteins, and can not be used for exploring the whole proteome. Furthermore, phenotypic changes can be caused for a multitude of reasons related to the gene mutations, and thus protein interactions suggested by experimental results would require confirmation at the biochemical level.
Bioinformatic Methods
Protein interactions can be investigated by using comparative genomics for the functional annotation of proteins. Currently, there are three major techniques. The first technique is called Domain Fusion (or Rosetta Stone), which assumes that protein domains are structurally and functionally independent units that can operate as discrete polypeptides. The second technique is based on the operon organization of bacterial genes, where such genes are often functionally related even if their actual sequences are disparate. The third technique uses phylogenic profiling, exploiting the evolutionary conservation of genes involved together in a particular function. Unfortunately, these bioinformatic methods require a complete genome sequence, and are generally limited to bacteria or other organisms with well-defined operons. Furthermore, the results are not conclusive evidence of specific protein interactions, and require confirmation at the biochemical level.
Affinity-Based Methods
Protein interactions can be investigated at the biochemical level by directly determining affinity between a protein and candidate interaction partners, such as in immunoassays. Proteins are immobilized onto a stationary phase or flat glass surface, and a mixture of potential complementary ligands is flooded over the immobilized protein. Binding is indicated by fluorescent or radioactive probes chemically attached to the ligands, which are then imaged. Unfortunately, protein functionality can be severely restricted by the immobilization process. A related technique chemically labels the proteins themselves and then floods them over a surface coated with immobilized ligands. However, this process suffers from the fact that proteins do not label uniformly with the same efficiency, and the chemical attachment of the labels can interfere with the range of the protein's interactions. Furthermore, attachment of labels can adversely affect protein solubility, and fluorescent probes may be quenched by the attachment. Detection may also be performed by electrochemical amperometry (e.g. U.S. Pat. No. 7,297,312), but the drawbacks remain.
Diffusion Immunoassays may use a pair of adjacent fluid flows in a microcapillary channel (e.g. U.S. Pat. No. 6,541,213, U.S. Pat. No. 7,271,007, U.S. Pat. No. 7,306,672, U.S. Pat. No. 7,060,446, U.S. Pat. No. 7,704,322, and U.S. patent applications US 2010/0263732, US 2008/0182273, US 2003/0096310, US 2009/0053732, and US 2003/0234356), or functional equivalents, where interactions between components in the two fluids at the flow interface causes a change in diffusion characteristics that affects the concentration profile near fluid interface. Detection of the concentration profile provides information on the interactions. This avoids complications associated with a stationary phase, but still prefers the use of labeling, and only one measurement per sample is practical it is non-cyclable). The use of multivalent reactants (e.g. U.S. Pat. No. 7,550,267) allows the use of components with a greater disparity of diffusion coefficients, but the measurement drawbacks of labeling and non-cyclability remain. Related devices using porous membranes (e.g. U.S. Pat. No. 5,212,065) suffer from the same disadvantages. The use of a thin polymer layer over an array of electrochemical sensors (e.g. U.S. Pat. No. 7,144,553) is capable of determining diffusion characteristics via time delays involved in permeating the polymer, but does not concentrate the analyte in a narrow hole (thereby enhancing sensitivity), and is not amenable to cycling the analyte towards and away from the sensor via hydrodynamic flow. Diffusion may be measured by optically tracking an analyte system (e.g. US 2008/0145856), but this has the drawback of preferring the use of labeling technology. Diffusion may be measured by detection of penetration depth into a hydrogel (e.g. US 2006/0115905), but this has the drawback of preferring the use of labeling technology.
Microchannel Conductometry measures changes to transverse conductance as protein molecules pass through a microchannel, and this has been described as possibly useful for label-free protein interaction detection (e.g. US 2005/0109621). However, that method only indirectly determines diffusion properties, and is not amenable cycling the measurements. Conductometry has also been used for label-free cell culture monitoring (e.g., U.S. Pat. No. 7,732,127 and U.S. Pat. No. 7,192,752), but these are not direct measurements of proteins and their interactions. The use of nanogaps US 2005/0136419) avoids certain double-layer complications of electrochemical measurements, but has the drawback of preferring the use of tethering technology.
Physical Methods
Protein interactions can be investigated at the physical level. The techniques of X-ray crystallography and nuclear magnetic resonance (NMR) determine the locations of protein atoms within the molecule, and the resulting 3-dimensional map can be used to suggest which other molecules are likely to fit into its topology and charge distribution. Unfortunately, X-ray crystallography requires the growth of protein crystals for each protein to be investigated, which is a difficult and time consuming process, and the crystal environment is drastically different than the aqueous environment in which the protein functions. NMR requires a large quantity of purified protein, and analysis of the resulting complex data can be inconclusive.
The technique of surface plasmon resonance uses protein adherence to metal films, but this can adversely affect protein functionality.
The technique of Fluorescence Resonance Energy Transfer (FRET) takes advantage of energy transfer that can occur between nearby fluorophores when the emission spectrum of one fluorophore overlaps the excitation of the other fluorophore. By labeling one candidate interaction partner with one fluorophore, and the other candidate interaction partner with another fluorophore, then interactions will be indicated by an increase in the fluorescence of one fluorophore at the expense of the other. This works well with even transient interactions. Unfortunately, this requires chemical attachment of a fluorophore to every protein, which may adversely affect protein functionality.
The technique of atomic force microscopy of dendron-isolated analytes (e.g. U.S. Pat. No. 6,645,558, US 2008/0113353, US 2009/0048120 and US 2010/0261615) can detect individual analyte molecules, but requires tethering bonds and extensive sample preparation.
Standard Expression Libraries
Protein interactions can be investigated through the use of libraries of cDNA that produce bait proteins that can be labeled and used as a probe. Typically, the bait proteins are produced through the use of phage particles. The technique allows for the association of as bait protein with its corresponding cDNA, but suffers from the major drawback of as low throughput; screenings for each bait protein are required. Furthermore, the production of the bait proteins is not under native conditions, leading to possibly erroneous folding and false negatives.
Phage Interaction Display
Protein interactions can be investigated through the use of an expression cloning strategy. A cDNA sequence is inserted into a phage protein coat gene, and cultured in bacterial cells. The phage than expresses as new protein on its coat, which then can be used for protein interaction analyses. If a mixture of such phages interacts with an immobilized labeled protein in a well, the well can be rinsed to leave behind only the interacting phages, along with the cDNA sequences that formed them. The cDNA sequences in turn can then be massively amplified by bacterial infection. This technique is highly amenable to automated parallel screenings. Unfortunately, as with standard expression libraries, the proteins are not formed under native conditions. Also, the technique is limited to short peptides that can be formed on the phage surface.
Yeast Two-Hybrid System
Protein interactions can be investigated through the use of transcription factors within yeast cells, which is a more native environment for protein expression than in vitro. A protein under investigation is expressed in a haploid yeast cell as a fusion with the DNA-binding domain from a transcription factor. Another protein is expressed in another haploid yeast cell as a fusion with the transactivation domain of the same transcription factor. Mating the two yeast strains into a diploid strain allows the two proteins to interact. If they do interact, the transcription factor will be assembled, causing a test gene to be activated. The technique is amenable to large-scale screenings, but there are several drawbacks. Experimental repeatability is quite low, suggesting inordinate sensitivity to environmental conditions, or that the screens were not comprehensive. There are a significant number of failures to detect interactions well-established from other more specific techniques, indicating high level of false negatives. Lastly, a significant number of detected interactions are determined to not be valid by further analysis, indicating a high level of false positives.
All publications referred to herein are hereby incorporated by reference in their entirety to the extent not inconsistent herewith.